title: Effects of the gout-causing Q141K polymorphism and a CFTR ΔF508 mimicking mutation on the processing and stability of the ABCG2 protein.
abstract: ABCG2 is an important multidrug transporter involved also in urate transport, thus its mutations can lead to the development of gout and may also alter general drug absorption, distribution and excretion. The frequent ABCG2 polymorphism, Q141K, is associated with an elevated risk of gout and has been controversially reported to reduce the plasma membrane expression and/or the transport function of the protein. In the present work we examined the stability and cellular processing of the Q141K ABCG2 variant, as well as that of the ΔF142 ABCG2, corresponding to the ΔF508 mutation in the CFTR (ABCC7) protein, causing cystic fibrosis. The processing and localization of full length ABCG2 variants were investigated in mammalian cells, followed by Western blotting and confocal microscopy, respectively. Folding and stability were examined by limited proteolysis of Sf9 insect cell membranes expressing these ABCG2 constructs. Stability of isolated nucleotide binding domains, expressed in and purified from bacteria, was studied by CD spectroscopy. We find that the Q141K variant has a mild processing defect which can be rescued by low temperature, a slightly reduced activity, and a mild folding defect, especially affecting the NBD. In contrast, the ΔF142 mutant has major processing and folding defects, and no ATPase function. We suggest that although these mutations are both localized within the NBD, based on molecular modeling their contribution to the ABCG2 structure and function is different, thus rescue strategies may be devised accordingly.
authors.affiliation: MTA-SE Molecular Biophysics Research Group, Budapest, Hungary.
authors.forename: Hajnalka
authors.initials: H
authors.lastname: Sarankó
authors.forename: Hedvig
authors.initials: H
authors.lastname: Tordai
authors.forename: Ágnes
authors.initials: Á
authors.lastname: Telbisz
authors.forename: Csilla
authors.initials: C
authors.lastname: Özvegy-Laczka
authors.forename: Gábor
authors.initials: G
authors.lastname: Erdős
authors.forename: Balázs
authors.initials: B
authors.lastname: Sarkadi
authors.forename: Tamás
authors.initials: T
authors.lastname: Hegedűs
mesh_terms: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000235:genetics*; D002462:Cell Membrane / Q000378:metabolism; D002942:Circular Dichroism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D005434:Flow Cytometry; D020022:Genetic Predisposition to Disease; D006073:Gout / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D009363:Neoplasm Proteins / Q000235:genetics*; D010654:Phenylbutyrates / Q000494:pharmacology; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D011499:Protein Processing, Post-Translational; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D059748:Proteolysis / Q000187:drug effects; D013696:Temperature
chemical_list: C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D050505:Mutant Proteins; D009363:Neoplasm Proteins; D010654:Phenylbutyrates; C093349:cystic fibrosis transmembrane conductance regulator delta F508; D019005:Cystic Fibrosis Transmembrane Conductance Regulator
keywords: 3R; 4-phenylbutyrate; ABCG2; CFTR; Cystic fibrosis; G188E, R191Q, and R193K rescue mutations in ABCG2-NBD; Gout; HEK; MBP; NBD; PBA; Polymorphism; SNP; Sf9; Spodoptera frugiperda cells; Stability; Structure; TMD; cystic fibrosis transmembrane conductance regulator, ABCC7; human embryonic kidney 293 cells; maltose binding protein; nucleotide binding domain; single nucleotide polymorphism; transmembrane domain